The present invention relates to the general field of devices and methods for performing flow measurements to characterize microparticles, and in particular biological cells. The present invention relates more specifically to measuring the refringence of biological microparticles together with other parameters that are useful in biomedical applications.
In this context, most automatic cytology analyzers, such as hematology analyzers, are designed to measure cell characteristics that enable a laboratory physician to identify the origins of certain disorders. For example, diagnosing certain anemias, certain cancers, or following up certain medicinal treatments, are all performed by observing physical characteristics of each cell.
The object of the present invention is to develop a device and a method enabling each of the populations of particles contained in a biological sample, and in particular in blood, to be better distinguished and thus better counted. The invention also makes it possible to measure very accurately the intracellular hemoglobin content of each population of erythrocytes. By extension, the device and the method of the invention enable the populations of leukocytes in the sample to be better analyzed.
Concerning the erythrocyte cell line, the routine parameters provided by the most common analyzers are the following: the number of red blood cells per unit volume (#RBC), the mean cell volume (MCV, conventionally given in cubic micrometers (μm3)), the red cell distribution index (or width) (RDW in %), the hematocrit level (HT in %), the mean hemoglobin concentration in the primary sample (HB), the mean corpuscular hemoglobin concentration (MCHC in grams per deciliter (g/dL)), and the mean corpuscular hemoglobin (MCH expressed in picograms (pg)).
Most hematology analyzers measure the parameters: #RBC, HB, and hematocrit level, with all of the other parameters being obtained by calculation.
Nevertheless, it should be observed that the most immature red blood cells, which contain residues of ribonucleic acid (RNA) and are known as “reticulocytes”, can be measured using certain types of analyzer. A dye or fluorescent marker is then used as a stain to reveal the presence of intra-cytoplasmic RNA. The fraction of red blood cells that express a fluorescence or diffraction signal that is specific to RNA marking serves to determine the number of reticulocytes per unit volume (#RET).
In addition to these parameters, diagnosing certain anemias or tracking their treatment requires knowledge of the corpuscular hemoglobin concentration (CHC) of each red blood cell and/or of each reticulocyte (CHCr: corpuscular hemoglobin concentration of reticulocytes). Unlike the parameters MCHC and MCH, which are mean values calculated over an entire population of red blood cells, CHC and CHCr are values for the concentration of corpuscular hemoglobin in each blood cell and they are used to construct histograms showing the distribution of hemoglobin over populations of red blood cells and the immature fringe (the reticulocytes). For example, two patients having identical mean corpuscular hemoglobin concentrations may present statistical distributions that are very different over the red blood cells and the reticulocytes. That is well known in cytology. Thus, when a smear is examined using a microscope, the color of a red blood cell is strongly correlated to its hemoglobin content: the greater the concentration of hemoglobin, the greater the attenuation of light. That observation is a consequence of the Beer-Lambert law that shows that optical density is proportional to the concentration of the solute. Knowledge about the statistical distribution of hemoglobin within different cells provides information about the state of health of a patient. Departure from normality is a symptom to be interpreted by a laboratory physician. For example, red blood cells having a low hemoglobin content are said to be “hypochromic”, while red blood cells having a high hemoglobin content are said to be “hyperchromic”. When the hemoglobin distribution varies strongly from one red blood cell to another, that is a symptom of polychromatophilia. Cells for which the content is abnormal often present rheological properties that are abnormal and that give rise to other physiological symptoms, in particular with thalassemias or drepanocytoses. The hematological interpretation of these parameters on healthy subjects and patients is set out in the article published in Blood and entitled “Simultaneous measurement of reticulocytes and red blood cell indices in healthy subjects and patients with microcytic and macrocytic anemia”, Onofrio et al., 1995, Vol. 85, pp. 818-823.
The hemoglobin content of reticulocytes is thus presently recognized as being a parameter that is very useful for diagnosing central anemias or indeed for following up the response of a patient to treatments. Furthermore, numerous publications in peer review journals and in numerous patent applications have been published over recent years, both about the clinical interpretation of hemoglobin distribution over all red blood cells, and about methods to be implemented in order to obtain that information.
For example, iron-deficiency anemias involve various fringes of the population, and in particular people under dialysis for whom an iron load balance is performed regularly. People suffering from hemorrhages or bleeding, people being treated by hemodialysis, or indeed people suffering from malnutrition, are liable to suffer from iron-deficiency anemias. Furthermore, subjects treated with erythropoietin (EPO) may develop an iron-deficient anemia syndrome even when their tissue reserves of iron are sufficient or even excessive, since the mechanism for synthesizing hemoglobin is limited by the deregulation of the iron fixing metabolism. Assays of ferritin and of the transferrin saturation coefficient are often used in diagnosing those biological disorders, but it is recognized that the serous concentration of those specific proteins is influenced by other factors in the event of inflammatory syndrome or infection.
The article entitled “Reticulocyte hemoglobin content (CHr): early indicator of iron deficiency and response to therapy” (Brugnara et al., Blood 1994, Vol. 83, pp. 3100-3101) teaches that the response to supplementary iron taken orally can be followed up by measuring the absolute count of reticulocytes and the corpuscular hemoglobin concentration. Thus, people who respond positively to that treatment benefit simultaneously from an increased number of reticulocytes and an increase in hemoglobin load from the first day. However, the mature red blood cell indices are not impacted until a very long time after the treatment, usually only after several weeks. Measuring #RET and the CHCr histogram is thus very important in determining in advance whether treatment is effective.
Thus, knowing the statistical distribution of the hemoglobin concentration over the red blood cell line, or indeed over a fraction thereof, is most advantageous in association with other more conventional parameters available from most hematology analyzers.
Concerning thrombocytes, it is most advantageous for the doctor or physician to be able to count and observe platelet activation, since that may represent an aggregation phenomenon that is at the origin of thromboses. This distinction between activated and non-activated platelets is relatively difficult to observe on a hematology analyzer.
The ability also to distinguish between platelets of large size (macroplatelets) and microcytes (which are red blood cells of very small size), is important in order to avoid making certain errors of diagnosis. Unfortunately, among hematology analyzers other than those based on lasers, there is at present no solution that is simple and effective for avoiding this potential confusion between macroplatelets and microcytes.
Concerning the leukocyte cell line, it is clear that accurately distinguishing between the various types of cell is a considerable advantage for enabling a laboratory physician to produce high-quality diagnoses. For example, the article “The cytoplasmic refractive index of lymphocytes, its significance, and its changes during active immunization (K. W. Keohane and W. F. Metcalf, Jan. 1, 1959, Experimental Physiology, 44, pp. 343-350) shows that measuring the refractive index of lymphocytes makes it possible to determine their level of activation after vaccination.
In order to measure the hemoglobin content of the red blood cell line, various methods based on flow cytometry have been proposed on the basis of optical measurements, mainly measurements of cell reflectivity or diffraction, such measurements being optionally combined with electrical measurements operating in continuous or pulsed manner via a measurement micro-orifice.
The first method of measuring corpuscular hemoglobin is attributed to D. H. Tycko. In U.S. Pat. No. 4,735,504, the author describes an optical method based on light diffraction measured in two regions of space that are identified by two parameters that correspond to very specific angles that are obtained by calculation. It should be observed that the red blood cell is previously treated chemically so as to give it a shape that is quasi-spherical. Within the limits of that approximation, it is possible to consider a red blood cell as being a uniform sphere of radius R and of refractive index IDX. Applying Mie theory to that simplified red blood cell model enables the intensity that is diffused in each region of three-dimensional space to be determined and enables an optimum configuration to be determined in which iso-concentration and iso-volume curves do not intersect, but on the contrary define a set of unambiguous curves. The set of curves makes it possible to solve the inverse problem consisting in determining the volume of the sphere and its refractive index on the basis of two measurements of the light diffracted in the two sub-pupils of the detection system.
The same author recognizes the complexity of that optical method in U.S. Pat. No. 5,194,909, column 4, lines 39 to 57. Instead of making two measurements of diffracted light, D. H. Tycko proposes measuring the volume of the cell by the conventional Wallace Coulter technique that consists in measuring the variation of impedance that results from a biological cell passing through a micro-orifice. That electrical measurement is associated with a measurement of light diffracted in a single pupil of the optical system. Once more, Tycko determines an annular pupil on the basis of two angles (θL; θH) that serves to define a set of curves giving a single solution for the hemoglobin content of the red blood cell on the basis solely of the measurement of the light diffracted in the pupil of the optical system.
Starting from the observation that calibrating the methods proposed by Tycko is very difficult to implement, since it is not possible to perform calibration with conventional latex beads, the Russian Novossibirsk team developed an original concept enabling the microparticle diffusion indicator to be measured. That system is referred to as a “scanning flow cytometer (SFC)”. The angle between the light source, the microparticle, and the detector is a function of the movement of the microparticle in the fluid stream. Thus, the movement of the microparticle makes it possible to scan an angular sector that enables the diffractive light indicator to be recorded over a relatively large range of angles. Various members of that Russian team have applied an SFC to measuring the volume and the hemoglobin content of red blood cells.
In the article entitled “Calibration-free method to determine the size and hemoglobin concentration of individual red blood cells from light scattering” (Sem'yanov et al. Applied Optics, Vol. 39, No. 31, November 2000), the authors show that the diffraction pattern produced by red blood cells presents a series of maxima and minima. The positions and the amplitudes characteristics of those extrema are combined in order to lead to a calculation algorithm that enables the volume and the hemoglobin concentration of red blood cells to be deduced without performing a prior calibration operation.
In U.S. Pat. Nos. 5,817,519 and 6,025,201, it is necessary to use a laser in order to measure diffraction at small angles. The measurement is based on Mie theory and the principle that is used is thus similar to that used in the devices described by Tycko. Thus, in those documents, combining a diffraction measurement with acquiring another signal that may be an optical signal or an electrical single makes it possible to calculate the refringence of platelets only. Other cells cannot be distinguished by the electro-optical device that is described.
In US 2005/0134833 in the name of Beckman Coulter Inc., D. L. Kramer accepts that the methods based on measuring forward light diffraction suffer from error because they rely on cell refringence, which itself depends on the intra-cytoplasmic refractive index. He states that that refractive index may vary considerably as a function of solutes other than hemoglobin, in particular the presence of salts. He recommends a device based on a reflectivity measurement in order to avoid the inaccuracies of other systems. The operation of his device is applied to measuring red blood cell hemoglobin. The reflectivity measurement is combined with a cell volume measurement obtained by the technique based on variation in the impedance of a measurement micro-channel having a continuous current passing therethrough on the Coulter principle. Although the effectiveness of that device is not demonstrated, the author states that the reflectivity parameter is correlated with the hemoglobin content.
A non-optical approach is proposed in patent WO 97/26528 filed by the same company. In that patent, the corpuscular hemoglobin content is determined by performing two electrical measurements: one continuously and the other under pulsed conditions. Under continuous conditions, the outside volume of the cell is measured, whereas under pulsed conditions, the field interacts with the intra-cytoplasmic content that presents conductivity, which appears to be controlled by the concentration of hemoglobin.
It should also be observed that the above-mentioned inventions or publications relate only to measuring the hemoglobin content of red blood cells without any mention of reticulocytes.
In flow cytometry, it is possible to detect a reticulocyte only after specifically marking the ribonucleic acid as described for example in patent FR 2 759 166 filed by the Applicant. A study comparing various methods of detecting reticulocytes (“Reticulocyte enumeration: past & present”, Riley et al., Laboratory Medicine, Vol. 32, No. 10, October 2001) finds in particular that fluorescent methods provide the best performance. More specifically, the thiazole orange compound is found to be one of the best markers of nucleic acids (DNA and RNA). The dominance of that molecule stems from that fact that it absorbs the incident light strongly and that the fluorescent yield is very high after hybridizing with nucleic acids. That method is proposed in all Horiba Medical analyzers for automatically counting reticulocytes (“Automated reticulocyte counting and immature reticulocyte fraction measurement”, Lacombe et al., American Journal of Clinical Pathology, Vol. 112, No. 5, November 1999).
By way of example, U.S. Pat. No. 5,350,695 in the name of Miles Inc. describes a method of staining nucleic acids with oxazine 750 or with New Methylene Blue. Oxazine 750 causes nucleic acids to precipitate, thereby enabling them to be detected by an optical extinction measurement. The authors recognize that their invention relies on adapting an optical extinction measurement to the conventional above-described Tycko device. The absorption measurement is used exclusively for distinguishing immature cells, in particular reticulocytes, whereas the diffraction signals are used for calculating the globular indices constituted by the volume and the corpuscular hemoglobin content. In that patent, it is accepted that the absorption measurement needed for resolving immature cells is interdependent with the diffraction measurement. In order to reduce such interference, as described in example 3, columns 15 and 16, the authors propose a cell-to-cell correction method. Unfortunately, those calculations are complex and likely to be inaccurate since they need to decouple two optical phenomena of small amplitudes: firstly the refringence effect and secondly the absorption effect, those two effects being mixed together in the signal measuring optical extinction. A first limit on that correction method comes from the fact that the blood cell is assumed to be a perfect uniform sphere. Mie theory is used to calculate the proportion of the signal that is to be subtracted from the optical extinction signal. That calculation gives a new absorption value that can be attributed solely to the contribution of the dye and used to measure the quantity of nucleic acid and thus to determine the degree of maturity of the red blood cell.
FIG. 18 of document U.S. Pat. No. 5,350,695 compares the results of counting reticulocytes between the said method and the manual counting method. Although the correlation coefficient is not given, it can be seen that the measurements are highly dispersed and that the reticulocyte count is inaccurate below 2%. However, it is known that the mean lifetime of a red blood cell is 120 days: it can therefore be estimated that the red blood cell renewal rate is on average equal to 0.8% per day, which explains from a purely biological point of view the need to have a sensitive method that is capable of measuring well below 2%.
U.S. Pat. No. 5,360,739, still in the name of Miles Inc., describes a method of determining the corpuscular hemoglobin content that is based on Tycko's principle, i.e. light diffraction is measured at two diffraction angles respectively referred to as “red scatter low” and “red scatter high”, whereas reticulocyte measurement is based not on measuring optical absorption as in U.S. Pat. No. 5,350,695, but on the principle of fluorescence. The authors envisage using two photo-excitable compounds, one in the red (oxazine 750) and the other in the blue (acridine orange and derivatives thereof). Under such circumstances, the authors propose coupling two lasers upstream from the vessel so that the light beams coincide at the measurement point. The proposed optical set-up uses illumination and detection axes that coincide, thereby introducing a considerable degree of complexity since, when using the compound that is photo-excitable in the blue, the set-up leads to making use of four measurement channels. In that method, an all-optical approach is preferred for determining the parameters of interest (volume and corpuscular hemoglobin concentration) of each red blood cell and of each reticulocyte contained in a blood sample.
U.S. Pat. No. 6,630,990 in the name of Abbott addresses the problem of measuring corpuscular hemoglobin concentration in a manner that is substantially equivalent to the above-described method. That patent presents an optical device enabling a plurality of cell lines to be resolved, in particular white blood cell lines and red blood cell lines. The system has a single light source and a single receiver presenting three measurement zones that define a singular configuration for using Mie theory to calculate the corpuscular hemoglobin and the cell volume. The main characteristics rely on using three diffraction angles for calculating the volume parameter V and the corpuscular hemoglobin HC, thereby leading to considerable complexity from a technological point of view, since a specific detector is necessary and the data is processed by a method operating in three-dimensional or four-dimensional space if fluorescence is added as a parameter for measuring the maturity of red blood cells. The complexity of the method of calibrating the device is also an important question that is not addressed in that document.
The parameter RET-y has been proposed by Sysmex for the XE2100 analyzer. Various experimental studies have been carried out by several teams in order to evaluate the clinical advantages of this RET-y parameter. For example, the article “New red cell parameters on the Sysmex XE-2100 as potential markers of functional iron deficiency” (Briggs et al., 2001, Sysmex Journal International, Vol. 11, No. 2) shows that the parameter RET-y is correlated with the hemoglobin content of reticulocytes. That parameter, which is the mean value of the forward scatter (FSC) restricted to the reticulocyte population only is calculated from the two-parameter FSC×FLUO representation of a sample of cells analyzed by flow cytometry in which, conventionally, the parameter FSC corresponds to the diffraction at small angles and the parameter FLUO corresponds to the fluorescence of a polymethine that is photo-excitable in the red and that is used for marking and quantifying nucleic acids (DNA/RNA). More recently, in the article “Potential utility of Ret-Y in the diagnosis of iron-restricted erythropoiesis” (Franck et al., Clinical Chemistry 50: pp. 1240-1242, 2004) C. Briggs et al. describe similar results. The authors recognize that the parameters MCH and CHr are correlated with the parameter RET-y. A non-linear regression method is also proposed that enables the Sysmex XE2100 analyzer to be calibrated with the reference Advia 120 analyzer under the Bayer trademark. Those results are described and set out in patent EP 1 967 840 A2 filed by Sysmex in March 2007. Unfortunately, the non-linearities of the response of the FSC can lead to considerable inaccuracy, in particular for high values of the parameter CHr. In particular, for values close to 40 pg, it can be seen that the method is not sensitive since RET-y varies very little around that value. Furthermore, that patent makes reference only to reticulocyte populations, the FSX×FLUO two-parameter representation not being adapted to measuring other particles in circulating blood, in particular leukocytes.
Measuring diffraction at small angles, as described in the prior art patents, e.g. the Tycko device and set-ups derived therefrom, takes place through an annular pupil of apparent diameter lying in the range 5° to 15°. In order to measure diffraction at small angles with a good signal-to-noise ratio, those set-ups make use of a laser. Under such operating conditions, the numerical aperture of the illuminating beam is substantially zero. From a practical point of view, using a laser is the only solution that makes it possible, with a numerical aperture that is substantially zero, to produce the radiation at high power (several milliwatts) needed for effective measurement of diffraction at small angles.
Devices based on the measurement principles described in the prior art are therefore expensive because they include a laser.
Furthermore, they require the use of a stopper for stopping the laser beam in effective manner. Since the stopper needs to be aligned with great accuracy in order to avoid saturating the detector, it is particularly difficult to fabricate and adjust devices operating on that principle.
Furthermore, prior art devices are very sensitive to the quality of the sphering of the blood cells by the reagent. In devices operating with diffraction at small angles, the diffraction phenomenon corresponds substantially to that of a plane wave, which, by definition, presents zero divergence. The nature of that interaction with a microparticle is very sensitive to the effects of the shape of the microparticle. Furthermore, when a microparticle is not a perfect sphere, the result of the measurement includes significant errors. This has been found experimentally, as shown in FIGS. 1A and 1B. These are two-parameter plots of volume (RES) and laser diffraction (FSC) parameters concerning red blood cells measured over angles in the range 1° to 30°. FIG. 1A was obtained with a diluant from the supplier ABX (patent FR 2 759 166—Fluored®) that possesses a weak sphering index, whereas FIG. 1B was obtained using the same system but with a reagent that possesses a high sphering index (the Bayer diluant used on the Advia 2120). It can be seen that FIG. 1A includes significant errors since the set of populations are highly dispersed. In contrast, in FIG. 1B, the populations of particles are situated in a region of the plane that defines a set that is more compact with a high degree of FSC×RES correlation as shown by the elliptical shape of the set.
It is therefore clear that, independently of the measurement system, the nature of the reagent has a considerable influence on the result in prior art devices.